Derivatization

Derivatization is primarily performed to modify an analytes function ality to enable chromatographic separations. For more than 40 years, Regis has been a leader in the manufacture of highly pure derivatization reagents for gas chromatography. The formation of chemical derivatives to facilitate meaningful analysis has long been a common practice in gas chromatography. For the analytical chemist, judicious use of derivatization can be the key to unlocking and simplifying a great many complex and perplexing separation problems. Derivatization, accomplished through alteration of functional groups, provides: Increased sample volatility Improved selectivity and chromatographic efficiency Enhanced detectability Additional procedures and references are available on our Web site at: www.registech.com/gc.

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Gas Chromatography (GC) Derivatization

Sample volatility or thermal stability is crucial in GC applications. If a sample does not possess these important characteristics, GC analysis is highly unproductive. Derivatization techniques have been developed to address these issues to insure successful separations. In GC Derivatization, replacement of active hydrogen in functional groups, such as -COOH, -OH, -NH, and -SH, is the primary area of concern and is accomplished through either silylation, acylation or alkylation.

Silylation
Silylation is the most widely used derivatization procedure for sample analysis by GC. Silylation reagents are popular because they are easy to use and readily form derivatives. In silylation, an active hydrogen is replaced by an alkylsilyl group, such as trimethylsilyl (TMS) or t-butyldimethylsilyl (t-BDMS). Compared to their parent compounds, silyl derivatives are more volatile, less polar, and more thermally stable. As a result, GC separation is improved and detection is enhanced. Silylation reagents are generally moisture sensitive, requiring them to be sealed under nitrogen to prevent deactivation. The derivatives of TMS reagents are also moisture sensitive. In response to this difficulty, t-BDMS reagents were introduced, which enabled the formation of derivatives 10,000 times more stable to hydrolysis than the TMS ethers. Both TMS and t-BDMS reagents are suitable for a wide variety of compounds, offer excellent thermal stability and can be used in a variety of GC conditions and applications. Analysis by the popular combination of gas chromatography and mass spectrometry (GS/MS) often requires special sample derivatization. Particularly effective in these applications is MTBSTFA.

Acylation
Acylation reagents offer the same types of advantages available from silylation reagents: creating less polar, more volatile derivatives. However, in comparison to silylating reagents, the acylating reagents more readily target highly polar, multi-functional compounds, such as carbohydrates and amino acids. In addition, acylating reagents provide the distinct advantage of introducing electroncapturing groups, thus enhancing detectability during analysis. Generally, these reagents are available as acid anhydrides, acyl derivatives, or acyl halides. The acyl halides and acyl derivatives are highly reactive and are suitable for use where steric hindrance may be a factor. Acid anhydrides are supplied in a number of fluorinated configurations, which improve detection. These fluorinated anhydride derivatives are used primarily for Electron Capture Detection (ECD), but can also be used for Flame Ionization Detection (FID). Fluorinated anhydrides are often used in derivatizing samples to confirm drugs of abuse. Despite the special utility of these reagents, their acidic nature requires that any excess or byproducts be removed prior to analysis to prevent deterioration of the column.

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Gas Chromatography (GC) Derivatization

Alkylation
As with other derivatization reagents, alkylation reagents reduce molecular polarity by replacing active hydrogens with an alkyl group. These reagents are used to modify compounds having acidic hydrogens, such as carboxylic acids and phenols. Alkylation reagents can be used alone to form esters, ethers, and amidesor they can be used in conjunction with acylation or silylation reagents. A two-step approach is commonly used in the derivatization of amino acids, where multiple functional groups on these compounds may necessitate protection during derivatization. Due to the availability of reagents and their ease of use, esterification (the reaction of an acid with an alcohol in the presence of a catalyst to form an ester) is the most popular method of alkylation. Alkylation reagents are available in several configurations that enable the formation of a variety of esters. Alkyl esters are stable, and can be formed quickly and quantitatively. By altering the length of the substituted alkyl group, retention of the derivative can be varied. In addition to the formation of simple esters, alkylation reagents can be used in extractive procedures where biological matrices may be present.

GC Chiral Derivatization
GC analysis of enantiomeric compounds on nonracemic or achiral stationary phases requires the use of enantiopure derivatization reagents. These reagents generally target one specific functional group to produce diastereomers of each of the enantiomeric analytes. From the resulting chromatograms, calculations are conducted to determine the enantiomeric concentration of the analyte.

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Guide to GC Derivatization Methods/Functional Groups

GC Derivatization Method
Functional Group Active Hydrogens Silylation BSTFA, BSTFA/TMCS, Deriva-Sil, Hydrox-Sil, TBH MSTFA, MTBSTFA, TMSI BSTFA, Hydrox-Sil Conc., MTBSTFA, TMSI Acylation PFPOH/PFPA Alkylation DMF Dialkylacetals,

Carboxylic Acids

PFPOH/PFPA

BF3/Methanol, BF3/n-Butanol, DMF Dialkylacetals, TBH DMF Dialkylacetals, PFB-Br/TBA-H-SO4, TBH DMF Dialkylacetals, PFB-Br/TBA-H-SO4, TBH DMF Dialkylacetals, TBH

Alcohols and Phenols: unhindered and moderately hindered Alcohols and Phenols: highly hindered

BSA, BSTFA/TMCS, HMDS, MTBSTFA/t-BDMCS

HFBI, Fluorinated anhydrides (HFBA, PFPA, TFAA), MBTFA, MCF* Fluorinated anhydrides (HFBA, PFPA, TFAA), HFBI, PFBCl Fluorinated anhydrides (HFBA, PFPA, TFAA), HFBI, MBTFA, PFBCl, TPC* HFBI

BSTFA/TMCS, Deriva-Sil, Deriva-Sil Conc.

Amines: primary and secondary

BSTFA, MTBSTFA/t-BDMCS

Amides

BSA, BSTFA, BSTFA/TMCS, Deriva-Sil Conc. BSTFA, TMSI

DMF Dialkylacetals,

Amino Acids

HFBI (+ Silylation)

DMF Dialkylacetals, TBH 3N HCl in n-Butanol

Catecholamines

TMSI

Fluorinated anhydrides (HFBA, PFPA, TFAA), HFBI MBTFA

Carbohydrates and Sugars Inorganic Anions Nitrosamines Sulfonamides

HMDS, Hydrox-Sil AQ, TMSI BSTFA, MTBSTFA

HFBA BSTFA Fluorinated anhydrides (HFBA, PFPA, TFAA) DMF Dialkylacetals, PFB-Br/TBA-H-SO4
Source: Knapp, D.R. Handbook of Analytical Derivatization Reactions; John Wiley and Sons: New York, 1979.

Derivatization reagents are listed in alphabetical order, not in order of preference.

*For Chiral Analysis

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Silyation Reagents

BSA
N,O-Bis(trimethylsilyl)acetamide Forms highly stable TMS derivatives with most organic functional groups under mild reaction conditions.

Product BSA

Size 10 x 1 4x5 25 100 gram gram gram gram

Catalog # 270501 270502 270503 270504

O

O H3C C

TMS N TMS
+

TMS

R + H3C

N H

TMS

TMS= Si(CH3)3

Y = O, S, NH, NR , COO R, R1 = Alk, Ar

BSTFA-Regisil BSTFA +TMCS (1%, 10%)

N,O-Bis(trimethylsilyl)trifluoroacetamide Reacts faster and more completely than BSA due to presence of trifluoroacetyl group. The high volatility of BSTFA and its byproducts results in separation of early eluting peaks. Highly volatile and stable products result in low detector noise and fouling. Excellent solubility. Addition of TMCS catalyzes reactions of hindered functional groups in secondary alcohols and amines.

Regisil RC-1 BSTFA

10 x 1 4x5 25 100 1000 10 x 1 4x5 25 100 1000 10 x 1 4x5 25 100 1000

+

gram gram gram gram gram gram gram gram gram gram gram gram gram gram gram
TMS

270111 270112 270113 270114 270116 270121 270122 270123 270124 270126 270131 270132 270133 270134 270135
Y R + F3C

Regisil RC-2 BSTFA +1% TMCS

Regisil RC-3 BSTFA +10% TMCS

O H3C C

TMS N TMS

O C N H TMS

TMS= Si(CH3)3

Y = O, S, NH, NR , COO R, R1 = Alk, Ar

HMDS
Hexamethyldisilazane Weak TMS donor, used for silylation of carbohydrates. Used as mixture with pyridine and trifluoroacetic acid.

HMDS
CH3 H3C Si N CH3 Si CH3 CH3
+

25 gram 100 gram

H Y R CH3 H3C Si CH3 Y R

270651 270652

CH3 H

CH3
+

Y = O, S, NH, NR1, COO R, R1 = Alk, Ar

H2N

Si CH3

CH3

MSTFA
N-Methyltrimethylsilyltrifluoroacetamide Most volatile of the TMS-acetamides. Useful in the analysis of volatile trace materials.

MSTFA

O F3C C N CH3
+

10 x 1 10 25 100
H Y R

gram gram gram gram

TMS

270590 270589 270593 270594

Y R + F3C

O C N H CH3

TMS TMS= Si(CH3)3

Regisil is a registered trademark of Regis Technologies, Inc.

Y = O, S, NH, NR1, COO R, R1 = Alk, Ar

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Silyation Reagents

MTBSTFA MTBSTFA + 1% t -BDMCS

N-Methyl-N-(t-butyldimethylsilyl) trifluoroacetamide Replaces active hydrogens to form t-BDMS derivatives. Derivatization is usually complete upon dissolution with this exceptionally strong, yet mild silylating reagent. MTBSTFA derivatives are 104 times more stable to hydrolysis than their corresponding TMS derivatives. Produces easily interpreted mass spectra for GC/MS. Addition of t-BDMCS catalyzes reactions of hindered alcohols and amines.

Product MTBSTFA + 1% t-BDMCS

Size 5 x 1 gram 10 x 1 gram 2 x 5 gram 25 gram 5 x 1 gram 2 x 5 gram 25 gram

O N C CF3
+

Catalog # 270141 270144 270142 270143 270241 270242 270243

R O Y R
+

MTBSTFA no t-BDMCS
CH3 CH3 H3C C Si

CH3 CH3 CH3 H3C

CH3 CH3 C Si H3C N H

Y = O, S, NH, NR1, COO R, R1 = Alk, Ar

CF3

CH3 CH3

TMCS
Trimethylchlorosilane Used as a catalyst to increase reactivity of other silylation reagents.

TMCS
CH3 H3C Si CH3 Cl
+

25 gram 100 gram

CH3 H Y R
1

270601 270602

H3C

Si CH3

Y = O, S, NH, NR R, R1 = Alk, Ar

TMSI
Trimethylsilylimidazole Potent, selective TMS donor that reacts with alcohols and phenols but not amines or amides. Derivatizes wet sugar samples, hindered hydroxyl groups in steroids, and amino acids in fluorinated acylation reagents. Used in the preparation of dual perfluoroacyl and TMS derivatives.

TMSI

10 x 1 gram 5 gram 25 gram

CH3 CH3 N
+ H

270401 270402 270403

H3C

Si CH3

N R, R1 = Alk, Ar

H3C

Si CH3

R + H

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Silyation Formulations

Deriva-Sil
BSTFA:TMCS:TMSI:Pyridine (3:2:3:10) formulation. Derivatizes sterically-hindered compounds. Reacts with carbohydrates, hydroxyand keto-steroids, fatty acids, and some amines and amides. Derivatizations are complete in minutes.

Product Deriva-Sil BSTFA:TMCS: TMSI:Pyridine (3:2:3:10)

Size 10 x 1 ml 25 ml

Catalog # 270151 270152

Deriva-Sil Concentrate
BSTFA:TMCS:TMSI (3:2:3) concentrate formulation. Used for applications where pyridine is undesirable (i.e., 3-ketosteroids).

Deriva-Sil Concentrate BSTFA:TMCS:TMSI (3:2:3)

25 ml

270150

Hydrox-Sil
HMDS:TMCS:Pyridine (2:1:10) formulation for one-step derivatizations. Fast formation of the TMS derivatives of organic acids, unhindered alcohols and phenols, and some amines.

Hydrox-Sil Reagent HMDS:TMCS:Pyridine (2:1:10)

25 ml

270457

Hydrox-Sil Concentrate
HMDS:TMCS (2:1) concentrate formulation. Suited for applications where pyridine in Hydrox-Sil is undesirable.

Hydrox-Sil Concentrate HMDS:TMCS: (2:1)

25 ml

270458

Derivatization Grade Solvents

Derivatization Grade Solvents
High purity reagents packaged under nitrogen. Sealed with Teflon-coated septa, allowing easy access to sample without exposure to moisture and oxygen.
Acetonitrile Pyridine 2 x 25 ml 2 x 25 ml 270010 270013

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Acylation Reagents

Fluorinated Anhydrides: HFBA/PFPA/TFAA

Heptafluorobutyric Anhydride/ Pentafluoropropionic Anhydride/Trifluoroacetic Anhydride Most commonly used for ECD. Reacts with alcohols, amines, and phenols. Bases such as triethylamine and trimethylamine can be added to promote reactivity. Frequently used for the confirmation of drugs of abuse. HFBA derivatives are the most sensitive to ECD. PFPA derivatives require the lowest analysis temperatures. TFAA is the most reactive and volatile of the anhydrides.

Product HFBA PFPA

Size 10 x 1 25 10 x 1 25 100 10 x 1 25 gram gram gram gram gram gram gram

O C3F7 + H Y R C3F7 C

Catalog # 270851 270853 640110 640113 640114 270841 270843

O Y R + C3F7 C OH

TFAA
HFBA C3F7 PFPA C2F5 TFAA F3C

O C O

O C

O C O

O C C2F5 + H Y R C2F5

O C Y R + C2F5

O C OH

O C O

O C CF3 + H Y R F 3C

O C Y R + F3C

O C OH

Y = O, NH, NR1 R, R1 = Alk, Ar

HFBI
Heptafluorobutyrylimidazole Readily forms derivatives with phenols, alcohols and amines suitable for ECD. Reactions are fast and mild. Imidazole is not acidic, so no decomposition or corrosion occurs on columns.

HFBI
O N N C C3F7
+

5 x 1 gram 5 gram
O H Y R C3F7 C

270611 270612

Y = O, S, NH, NR1 R, R1 = Alk, Ar

MBTFA
N-Methyl-N-bis(trifluoroacetamide) Reacts rapidly under mild conditions with primary and secondary amines. Reacts more slowly with alcohols, phenols, and thiols. Works well in the analysis of sugars.

MBTFA

10 x1 5 25 100

gram gram gram gram

270092 270091 270095 270093

PFPOH
2,2,3,3,3-Pentafluoropropanol Used in combination with PFPA to make derivatives of the most common functional groups, especially polyfunctional bio-organic compounds. Formed derivatives are highly suitable for ECD.

PFPOH

5 gram 25 gram
R1 O C OH PFPA

270815 270816
R1 C2F5H2C O C R2 O C CH2C2F5

C2F5H2C

OH + HO

C R2

R1, R2 = H, Alk, Ar

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GC Chiral and Specialty Derivatization Reagents

TPC
N-Trifluoroacetyl-L-Prolyl Chloride Couples with amines to form diastereomers which can be separated on GC columns. Provides sample volatility. Used for confirmation of drugs of abuse testing.

Product TPC
O N O C C CF3 Cl + H2N

Size 25 gram
R1 C R3 O R1, R2, R3 = H, Alk, Ar R2 N C

Catalog # 440001
O C CF3 N H R1 C R3 R2

MCF
(1R, 2S, 5R)-(-)-Menthylchloroformate Resolves enantioenriched alcohols.

MCF CH3

25 gram
CH3

440003

O O H3C CH CH3 C Cl + HO

R1 C R3 R2 H3C CH O CH3

O C O

R1 C R3 R2

R1, R2, R3 = H, Alk, Ar

HFIP
1,1,1,3,3,3-Hexafluoro-2-Propanol Esterification reagent for the determination of aromatic acids in tissue by GC and electron capture detection.

HFIP

25 gram 100 gram

270702 270704

(R)-(-)-MTPA-Cl
Mosher's acid chloride (R)-(-)- -(trifluoromethyl) phenylacetyl chloride (R)-(-)-MTPA-Cl is a ready to use reagent for the determination of enantiomeric purity of alcohols and amines.

(R)-(-)-MTPA-Cl Mosher's acid chloride

100 mg 500 mg 1000 mg

270900 270901 270902

Teflon is a registered trademark of DuPont Company.

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GC Chiral and Specialty Derivatization Reagents

3.0 N HCL in n-Butanol

Most commonly used for rapid diagnosis of neonatal blood spots by Tandem Mass Spectrometry.

Product 3.0N HCl in n-Butanol

Size 4 x 25 ml 100 ml 500 ml

Catalog # 201007 201009 201010

(R)-(-)-MTPA-Cl

3N HCl in n-Butanol is a derivatization reagent required for newborn screening for metabolic disorders. Neonatal screening, which has become a standard procedure in many countries, measures amino acids and acylcarnitines from a single drop of blood. Blood concentration of one or several of these compounds is either abnormally high or low in a variety of metabolic disorders in newborns. Derivatization with 3N HCl in n-Butanol ensures butylation of the carboxyl acid group of the analyte and formation of butyl ester, which forces ionization or makes charging of the analytes more efficient. Although direct analysis of extracted acycarnitine without derivatization is possible, according to different reports, butylesterification is superior with regard to sensitivity and specificity. Methods that include derivatization with 3N HCl in n-Butanol is the only validated procedure at this time. Many factors contribute to the success of a newborn screening process. Any impurities in derivatization reagent can potentially interfere with the analysis. 3N HCl in n-Butanol from Regis Technologies is manufactured under cGMP protocols to assure highest quality and lot-to lot consistency for this reagent. Each lot is tested by tandem mass spectrometry to ensure absence of contaminants which may interfere with analysis. Our Quality Assurance department reviews and approves all production documentation and test results. Regis takes necessary precautions that assure the quality of our 3N HCl in n-Butanol.

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GC Chiral and Specialty Derivatization Reagents

Notes

For additional information on GC Derivativization Reagents, contact us by phone, 8 0 0 . 3 2 3 . 8 1 4 4 ext. 662 8 4 7 . 9 6 7 . 6 0 0 0 ext. 662 or e-mail, sales@registech.com
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Please note: Certain GC Derivatization products are hazardous chemicals and additional shipping and handling charges could apply.